P4HA2 promotes proliferation, invasion, and metastasis through regulation of the PI3K/AKT signaling pathway in oral squamous cell carcinoma

Proline 4-hydroxylase 2 (P4HA2) is known for its hydroxylase activity, primarily involved in hydroxylating collagen precursors and promoting collagen cross-linking under physiological conditions. Although its overexpression influences a wide variety of malignant tumors' occurrence and development, its specific effects and mechanisms in oral squamous cell carcinoma (OSCC) remain unclear. This study focused on investigating the expression patterns, carcinogenic functions, and underlying mechanisms of P4HA2 in OSCC cells. Various databases, including TCGA, TIMER, UALCAN, GEPIA, and K-M plotter, along with paraffin-embedded samples, were used to ascertain P4HA2 expression in cancer and its correlation with clinicopathological features. P4HA2 knockdown and overexpression cell models were developed to assess its oncogenic roles and mechanisms. The results indicated that P4HA2 was overexpressed in OSCC and inversely correlated with patient survival. Knockdown of P4HA2 suppressed invasion, migration, and proliferation of OSCC cells both in vitro and in vivo, whereas overexpression of P4HA2 had the opposite effects. Mechanistically, the phosphorylation levels of the PI3K/AKT pathway were reduced following P4HA2 silencing. The study reveals that P4HA2 acts as a promising biomarker for predicting prognosis in OSCC and significantly affects metastasis, invasion, and proliferation of OSCC cells through the regulation of the PI3K/AKT signaling pathway.


Patients and tissue samples
We collected 44 fresh frozen OSCC tissue samples from patients (mean age 63; 23 males and 21 females) who underwent surgery at Qingdao Municipal Hospital Affiliated to Qingdao University between 2015 and 2018.The clinicopathological features including lymph node metastasis, tumor grade, stage, gender, and age were recorded.All procedures were conducted with patient consent, and the study was approved by the Qingdao Municipal Hospital Affiliated to Qingdao University.

Cell culture and reagents
SCC-9 and SCC-25 cells, provided by Professor Zheng from the Department of Oromaxillofacial Head and Neck Oncology at Shanghai Ninth People's Hospital, China, were cultured in Dulbecco's Modified Eagle's Medium (DMEM, GIBCO, US) supplemented with 100 U/mL streptomycin, 100 U/mL penicillin, and 10% fetal bovine serum (FBS, Sciencell, US).Cells were maintained at 37 °C in a 5% CO 2 environment.When cells reached 80%-90% confluence, they were subcultured at a ratio of 1:4 for further experiments.Additionally, the P4HA2 enzymatic agonist Isosaponarin was purchased from MedChemExpress (HY-N2589, New Jersey, US).

Cell transfection and stable cell lines Construction
We transfected all cells with specific shRNA-P4HA2-Lentivirus to knockdown P4HA2 or scrambled shRNA-GFP-Lentivirus (Guangzhou Yuanjing Biotechnology Co., Ltd, Guangzhou, China) at a multiplicity of infection of 50 for 24 h in culture medium containing 5 mg/mL polybrene when the cells reached 30% confluence.Stable cell lines were selected using puromycin post-transfection.Additionally, Lipofectamine 3000 was diluted and mixed with plasmid vectors pcDNA3.1-p4HA2or pcDNA3.1 (Miaolingbio.lnc,Wuhan, China) according to the manufacturer's instructions, and then added to the culture medium for transfection.This experiment included the sh-P4HA2 group, the sh-GFP group, the pcDNA3.1-p4HA2group, and the pcDNA3.1 group.(Supplemental Table 1 lists the interference sequences).

qRT-PCR
Total RNA was extracted from a variety of cells using the TaKaRa MiniBEST Universal RNA Extraction Kit (TakaRa, Japan).Reverse transcription was performed using the PrimeScript RT Reagent Kit with gDNA Eraser (TakaRa, Japan).The expression level of P4HA2 was quantified by real-time PCR (qRT-PCR) using SYBR Premix EX Taq II (Takara, China) under the following thermal cycling conditions: 60 °C for 30 s, followed by 40 cycles of 95 °C for 5 s, and 95 °C for 30 s. Supplemental Table 2 lists the primer sequences 9 .mRNA levels were quantified using the Comparative threshold cycle (Ct) method and the 2 -∆∆Ct analysis method.

Cell invasion test
Add 20 μL of Matrigel (Becton-Dickinson, the US) diluted 1:8 (Matrigel:DMEM) into the BioCoat Corning Matrigel Invasion Chamber (8 μm pore size; Becton-Dickinson, the US).We placed the Transwell chamber containing Matrigel on a 24-well plate.The transfected cells (2 × 10 5 cells in 200 μL of medium) were placed in the upper chamber.In the lower chamber, we filled it with 500 μL of complete culture medium containing 10% FBS to serve as an attractant.After 48 h, we fixed the invaded cells with 4% paraformaldehyde and stained them with 0.1% crystal violet.The bottom membrane of the chamber was examined under a microscope at 200× magnification, and 5 random fields were selected for photography.The number of cells that had passed through the bottom membrane was recorded using ImageJ software, and the average value was calculated.The experiment was repeated three times.

Cell scratch test
We cultured all cells on a six-well plate and created scratches using a 20 μL pipette tip.PBS was used to wash away the floating cells three times.Cells were then cultured in DMEM medium without FCS.Furthermore, the distance between the two edges of the scratch was measured under the microscope 48 h later.We observed the scratch wound areas immediately and 48 h after the scratch under the microscope.Cell migration was quantified as the percentage of wound healing relative to the initial wound area.

Cell viability test
Seed all experimental cells at a density of 2 × 10 5 cells per well and culture the cells in medium supplemented with 10% FBS for 0, 24, and 48 h.Then, add 10 μL of CCK-8 to each well and incubate at 37 °C for 2 h.Afterwards, measure the optical density (OD) of each well using a microplate reader at 450 nm.After the measurement, return the 96-well plate to the incubator until the next test point.Each group had five replicates at each time point.

Cell cycle analysis and cell apoptosis test
All cells were seeded in a 6-well culture plate, with each well reaching a density of 2 × 10 5 cells and a total volume of 2 mL.The cells were then fixed using 70% cold ethanol at − 20 °C overnight.The fixed cells were washed twice with precooled PBS and stained with a solution containing propidium iodide (PI, 50 μg/mL; Sigma Aldrich, St. Louis, the US) and RNase A (100 μg/mL; Sigma Aldrich, St. Louis, the US) at 4 °C for 30 min.A flow cytometer (Beckman Coulter, the US) was used to quantify the DNA content of the cells.Data were obtained using FlowJo software (version V10; https:// www.flowjo.com/ solut ions/ flowjo/ downl oads).For the apoptosis test, cells were treated with 0.25% trypsin (without EDTA), centrifuged at 500 g for 5 min, and then subjected to staining with Annexin V-PE and/or PI for 15 min at room temperature.A FACSCalibur flow cytometer was used for analysis.

IHC staining
We fixed OSCC and adjacent tissues, followed by an embedding process as per standard protocols.The embedded tissues were sectioned into 4 μm slices and subjected to staining using a conventional immunohistochemistry procedure.To block endogenous peroxidase, sections were treated with hydrogen peroxide.The sections were then incubated overnight at 4 °C with P4HA2 antibody (1:200, Proteintech).Following this, sections were incubated with an anti-rabbit secondary antibody for 30 min at room temperature.Color development was achieved using 3-diaminobenzidine tetrahydrochloride (DAB reagent kit, ZLI-9017, ZSGB-BIO, China) and counterstained with hematoxylin.Staining intensity was evaluated under a microscope, identifying cells with brown cytoplasm as positive.Two pathologists blindly assessed the staining intensity of the slides without prior knowledge of the patients' basic information or clinical features.During high-power microscopic examination, five fields were selected, and the number of positive cells was counted to obtain an average.

Xenograft tumor model test
BALB/c nude mice (female, aged 4-6 weeks) were provided by Vital River Laboratory Animal Technology Co., Ltd.(Beijing, China).Mice were maintained under standard pathogen-free conditions and randomly assigned to two groups, each consisting of six mice.Each mouse was injected subcutaneously on the left side with 5 × 10 6 sh-P4HA2 or sh-GFP cells.Body weight and tumor size were monitored post-injection.Tumor size was measured every five days using calipers, and volume was calculated using the formula: V = 1/2 × A × B 2 , where A and B represent the tumor's largest and smallest diameters, respectively 15 .After 26 days, the mice were dissected, and tumors were stained with hematoxylin-eosin (H&E).For the pulmonary metastasis model, two groups of Vol:.( 1234567890 www.nature.com/scientificreports/BALB/c nude mice were injected via the tail vein with 1 × 10 6 sh-P4HA2 or sh-GFP cells.Four weeks later, lung tissues were harvested, fixed in formalin, and the lymph nodes were counted.Animal husbandry and experimental procedures were approved by the Ethics and Humanities Committee of Qingdao Municipal Hospital (Shandong, China) and were performed in full compliance with relevant ethical guidelines (Approval ID: 2,023,108).

H&E staining
Lung specimens and subcutaneous tumors were fixed in 4% paraformaldehyde, dehydrated with graded ethanol, cleared with xylene, infiltrated with wax, embedded in paraffin, sectioned, and routinely stained with H&E for microscopic observation (Olympus, BX61, Japan).

Statistical analysis
Data analysis was performed using SPSS (version 16.0; https:// www.ibm.com/ spss).Results are expressed as means ± standard deviation (SD).Multiple comparisons were conducted using one-way ANOVA followed by LSD as a post-hoc test.Differences between two groups were analyzed using the Student's t-test.Normal distribution was assessed using the Kolmogorov-Smirnov and Shapiro-Wilk tests.For non-normally distributed data, comparisons between two groups were made using the Mann-Whitney test, and among multiple groups using the Kruskal-Wallis test.Overall survival (OS) of patients with different P4HA2 levels (using median as cutoff) was compared using the Kaplan-Meier method and log-rank test.Non-significant differences were denoted as 'ns' .Statistical significance was considered at *P < 0.05; **P < 0.01; ***P < 0.001;****P < 0.0001.Each experiment was conducted three times.

P4HA2 achieves high expression in OSCC tissues in contrast to normal tissues
We utilized the UALCAN, TIMER, and TCGA databases to investigate P4HA2 expression in normal and OSCC tissues (Fig. 1A,B).The data suggest that, in contrast to normal tissues, P4HA2 expression is significantly higher in OSCC tissues.Additionally, the UALCAN database indicates a significant correlation between P4HA2 expression and clinical stage, pathological grades, and lymph node metastasis (Fig. 1C).We also conducted a function enrichment analysis using the Gene Ontology database (http:// geneo ntolo gy.org/) to explore the potential functions of P4HA2 in depth.The results show that P4HA2 is notably enriched in several pathways, including the Calcium signaling pathway, cytokine-cytokine receptor interaction, and PI3K/AKT signaling pathway (Fig. 1D).

P4HA2 might be a prognostic biomarker for OSCC
To validate our findings, we analyzed P4HA2 expression in 44 OSCC tissue specimens using qRT-PCR and IHC analysis.The IHC results show that positive P4HA2 protein expression is characterized by cytoplasmic staining with a brownish-yellow color.P4HA2 expression was negative in well-differentiated OSCC tissues, weakly positive in moderately differentiated tissues, and notably positive in poorly differentiated tissues, indicating a significant correlation with the degree of tissue differentiation (Fig. 2A).Furthermore, The relative expression of P4HA2 mRNA was found to be higher in OSCC tissues compared to adjacent normal tissues (Fig. 2B).Additionally, semi-quantitative scoring analysis revealed a significant correlation between P4HA2 expression and clinical stage, pathological grades, and lymph node metastasis.However, there was little association between P4HA2 expression levels in cancer tissues and patients' gender, age, or smoking history (Supplemental Table 3).Overall, our findings are consistent with the data obtained from the UALCAN database (Fig. 1C).For a more detailed examination of P4HA2's role in OSCC, we used the K-M plotter and GEPIA database to evaluate the prognosis of all OSCC patients.The data indicate that patients with P4HA2 overexpression have a poorer overall prognosis compared to those with low expression (Fig. 2C).Furthermore, high P4HA2 expression, along with OSCC grade and stage, significantly impacts patient survival, as evidenced by univariate analysis results.Multivariate analysis also shows that age, tumor stage, and high P4HA2 expression are correlated with overall survival (Fig. 2D and Supplemental Table 4).

P4HA2 silencing and overexpression models were constructed
We constructed models of P4HA2 silencing (sh-P4HA2 and sh-GFP) and overexpression (pcDNA3.1-p4HA2and pcDNA3.1)by transfecting these constructs into OSCC cells.Subsequent qRT-PCR and Western blot tests confirmed the successful construction of these models in SCC-25 and SCC-9 cell lines (Fig. 3A).

P4HA2 adjusts the OSCC cells' proliferation
To assess the impact of P4HA2 on the function of SCC-25 and SCC-9 cells, we performed both CCK-8 and apoptosis tests.The CCK-8 test results demonstrate that P4HA2 facilitates cell proliferation (Fig. 3B).Additionally, this study suggests that P4HA2 affects the cell cycle, specifically regulating the transition from G1/G0 to S phase, as shown by flow cytometry (Fig. 3C).Flow cytometry also indicates that inhibition of P4HA2 expression can accelerate cell apoptosis (Fig. 3D).Based on the results of the cell proliferation and apoptosis experiments, we further explored the related molecular mechanisms.The protein levels of Cyclin D1, a cell cycle regulator associated with the G1/G0 to S transition, and survivin, a protein associated with cell apoptosis, were analyzed through Western blot tests.The results indicate that P4HA2 knockdown suppresses the expression of Cyclin D1 and survivin, whereas P4HA2 overexpression leads to increased expression of these proteins (Fig. 3E).Thus, P4HA2 can influence cell apoptosis and proliferation through its regulation of the cell cycle via Cyclin D1 and survivin in OSCC cells.

P4HA2 adjusts OSCC cells' metastasis and invasion
We performed tests in vitro to analyze how P4HA2 affects the biological behavior of OSCC.We performed the transwell invasion test and the scratch test to analyze the influence of P4HA2 on OSCC cells' migrating and invading processes.The transwell invasion test results indicated that the scratch wound healing time was prolonged after silencing P4HA2 expression.After P4HA2 knockdown, transmembrane cells' number declined.However, P4HA2 overexpression reversed the results (Fig. 4A,B).The above-mentioned result suggested that P4HA2 can positively regulate OSCC cells' capability of migrating and invading.Likewise, we used WB tests to analyze the molecular mechanism involved in depth.Western blotting test was used to measure MMP-2 and MMP-9  expression, and OSCC cell molecular mechanism (i.e., migration and invasion) was investigated.The results indicated that silencing P4HA2 down-regulated MMP-9 and MMP-2 expression, while P4HA2 overexpression promoted MMP-9 and MMP-2 expression (Fig. 4C).In conclusion, P4HA2 could affect OSCC cells' capability of migrating and invading based on the regulation of MMP-9 and MMP-2 expression.

P4HA2 regulates the PI3K/AKT pathway of OSCC cells in a collagen-dependent manner
The PI3K/AKT signaling pathway is well-known in tumor proliferation, invasion, and metastasis.To verify whether P4HA2 could regulate the PI3K/AKT pathway to play biological roles in OSCC cells, we evaluated collagen I, collagen IV, P13K, p-P13K, AKT and p-AKT expression in OSCC cells.And the results of WB tests indicated that collagen I, collagen IV, p-P13K and p-AKT expression in OSCC cells were notably reduced in the sh-p4HA2 group, while those in the pcDNA3.1-P4HA2group were notably higher than those in the sh-p4HA2 group.Interestingly, PI3K and AKT expression did not change notably between the sh-p4HA2 group and the pcDNA3.1-P4HA2group (Fig. 5A,B).In general, P4HA2 expedites OSCC cells' proliferating, invading, and migrating processes based on the PI3K/AKT pathway.Moreover, to further test whether it is collagen that regulates the PI3K/AKT pathway, we utilized a P4HA2 enzyme agonist, Isosaponarin, to promote the synthesis of collagen in OSCC cells.Similar results were observed: both phosphorylated PI3K and AKT expressions, not the total protein expressions, were significantly increased by upregulating the biological function of the hydroxylase, implying that P4HA2 regulates PI3K/AKT signaling in a collagen-dependent manner (Fig. 5C).

P4HA2 adjusts OSCC tumors growth in xenograft model
We injected sh-P4HA2 and sh-GFP cells into the BALB/c nude mice for subcutaneous tumor formation to further explore the role of P4HA2 in OSCC growth.At the same time, the same two groups of cells were injected into other nude mice through the tail vein.The nude mice were sacrificed 6 weeks later, and the subcutaneous  tumors and lung tissues were removed.The experiment indicated that the average body weight of the sh-P4HA2 group mice was lighter than that of the sh-GFP group.The tumor volume of the sh-P4HA2 group was notably smaller than that of the sh-GFP group.There were fewer lymph nodes in the lungs of sh-P4HA2 mice than in sh-GFP mice.Furthermore, the data of H&E staining indicated that the sh-P4HA2 group had a lower nucleus atypia degree.The above-described results suggested that P4HA2 can facilitate the occurrence and development of OSCC in vivo (Fig. 6).

Discussion
OSCC is the most common type of oral cancer.Its rapid growth, strong invasiveness, and high rate of neck lymph node metastasis are significant reasons for the high mortality rate among patients 16 .Therefore, it is urgent to explore the molecular mechanisms affecting the proliferation, invasion, and metastasis of OSCC, and to identify biomarkers that can accurately predict prognosis and prevent recurrence, progression, and metastasis.P4HA2 has been reported as a crucial enzyme that plays a significant role in cells' collagen synthesis and mature collagen formation 17 .The extracellular matrix (ECM) provides the physical framework for cells and initiates key cellular events such as survival, differentiation, proliferation, migration, and adhesion 18 .Mature collagen serves as a scaffold for the ECM and is the main component of it 19 .Additionally, it provides support for cells in spatial structures and acts as an anchor point for cell mechanical sensing 20 .Thus, P4HA2 is significantly correlated with cell migration, proliferation, differentiation, and other biological behaviors, playing a vital role in these processes.Furthermore, numerous signaling pathways within its structure regulate cell behavior 21 .Existing research has suggested that P4HA2 plays a role in many biological processes, including collagen maturation, epithelial-mesenchymal transition, and the deposition of amyloid β in Alzheimer's disease, and is vital in promoting tumor progression [22][23][24] .In this study, the results indicated that P4HA2 expression in OSCC tissues was significantly higher than in adjacent tissues, and P4HA2 expression in cancer tissues showed a negative correlation with www.nature.com/scientificreports/ the degree of differentiation in OSCC tissues.Moreover, high P4HA2 expression is positively correlated with high stage, high grade, and high lymph node metastasis rate in OSCC.The survival time of patients with high P4HA2 expression was also shorter than that of those with low P4HA2 expression.Multivariate analysis indicated that high P4HA2 expression is an independent poor prognostic factor for OS.Additionally, expression levels of both type I and type IV collagen were significantly altered-decreased and increased, respectively-after P4HA2 was knocked down and overexpressed, and both collagen types were positively correlated with P4HA2 expression.These results suggest that P4HA2 is critical to the occurrence and development of OSCC and shows a close correlation with the survival and prognosis of OSCC patients, possibly relating to the biological function of collagen.Matrix metalloproteinase (MMP) is a highly homologous Zn 2+ endopeptidase secreted by tumor and stromal cells.MMP-2 and MMP-9, members of the matrix metalloproteinase family, play a vital role in various tumors and are considered key regulators of cancer progression 25 .They can affect cancer migration and invasion by regulating the proteolysis of the vascular basement membrane and opening the metastasis pathway for endothelial cells, which is critical to the invasion and metastasis of breast cancer, pancreatic cancer, and oral squamous cell carcinoma [26][27][28][29] .In this study, the data indicated that cell migration and invasion were suppressed after the downregulation of P4HA2 expression.However, overexpression of P4HA2 had the opposite effect.This phenomenon was also observed at the expression levels of MMP-2 and MMP-9.These experimental results suggest that P4HA2 affects the migration and invasion of OSCC cells through the regulation of MMP-9 and MMP-2 expression.However, it is necessary to further clarify how P4HA2 affects MMP-2 and MMP-9 and to explore the specific molecular mechanisms by which P4HA2 regulates these enzymes.
In this study, we utilized the Gene Ontology (GO) database to analyze the potential mechanisms of P4HA2.The results indicated that P4HA2 was not only significantly enriched in ECM-receptor interactions but also in the PI3K/AKT signaling pathway in head and neck squamous cell cancer.PI3K is a catalytically active intracellular signaling protein that can adjust the phosphorylation level of downstream AKT, thus constituting the PI3K/ AKT signaling pathway.This pathway plays a crucial role in controlling cell proliferation, growth, and survival and is activated in various cancers [30][31][32][33] .Phosphorylated PI3K and AKT may contribute to the biological behavior of cancer cells, such as proliferation, migration, and invasion.Studies have shown that the PI3K/AKT signaling pathway is abnormally activated in liver cancer, gastric cancer, breast cancer, oral squamous cell carcinoma, and other cancers, and participates in the development and progression of tumors.Additionally, it has been shown that changes in the conformation of ECM could lead to changes in the ambient pressure of cells, which activate transmembrane proteins such as integrins, phosphorylate intracellular focal adhesion kinase 2 (FAK2), activate proto-oncogene sarcoma receptor coactivator (Src), and eventually activate the PI3K/AKT signaling pathway to regulate the behavior of tumor cells 34 .The expression of PI3K/AKT signaling pathway-associated proteins was down-regulated after P4HA2 expression was inhibited, while P4HA2 pathway-associated protein expression was up-regulated after the promotion of P4HA2 expression.These results suggest that P4HA2 could regulate the PI3K/AKT signaling pathway in OSCC cells.Furthermore, the potential link between P4HA2 and the PI3K/ AKT signaling pathway was explored by a rescue assay using a collagen synthesis agonist.These results suggest that P4HA2 can regulate PI3K/AKT expression at the protein level, but not at the mRNA level.Additional results showed that PI3K/AKT could be upregulated by promoting the collagen hydroxylase activity of P4HA2, supporting our proposal that PI3K/AKT expression activation is via a P4HA2 enzyme-mediated protein-protein interaction rather than direct transcriptional activation.Therefore, the above results indicated that MMPs are the critical downstream effectors of the P4HA2-collagen-PI3K/AKT axis.The underlying mechanism may be regulating the collagen-dependent PI3K/AKT signaling pathway.
Abnormal tumor proliferation arising from cell cycle disorder is a key factor in tumorigenesis.As a key cell cycle regulatory protein, Cyclin D1 is located on chromosome 11q13, which can drive cells from the G1/G0 phase to the S phase 35 .Existing research has suggested that Cyclin D1 is frequently dysregulated in cancer and could be a vital biomarker for cancer phenotype and disease progression 36 .Numerous signaling pathways (e.g., Ras/MEK/ ERK, PI3K/AKT, and Ral) are capable of regulating Cyclin D1 expression.The protein degradation of Cyclin D1 is regulated by glycogen synthase kinase 3β (GSK-3β).After PI3K activates AKT, AKT phosphorylates GSK-3β to inactivate it, which can up-regulate Cyclin D1 protein expression 37 .Survivin, the smallest member of the inhibitor of apoptosis protein family, is a 16.5 kDa protein encoded by the BIRC5 gene on chromosome 17 38 .Additionally, it is critical in regulating cell division and blocking caspase activation to inhibit apoptosis.Compared with normal tissues, survivin is overexpressed in most human cancers, and its abnormal expression is correlated with tumor proliferation, apoptosis, angiogenesis, treatment resistance, and poor prognosis 39 .It has been reported that the survivin gene is not expressed in normal human oral keratinocytes but is highly expressed in four OSCC cell lines.Moreover, existing research has suggested that the abnormal expression of survivin in tumor cells is regulated by a variety of factors, including microRNA (miRNA), receptor tyrosine kinase (RTK), and their downstream signaling pathways (e.g., MAPK, PI3K/AKT, signal transducer, TAT3, and other pathways) 40 .In the experiments of this study, we found that Cyclin D1 and survivin protein expression was down-regulated after silencing P4HA2.Conversely, P4HA2 overexpression led to up-regulated Cyclin D1 and survivin expression.The above-mentioned results suggest that P4HA2 can play a certain role in the apoptosis and proliferation of OSCC cells through the regulation of Cyclin D1 and survivin via the PI3K/AKT signaling pathway.

Conclusion
In summary, this study indicates that P4HA2 is highly expressed in OSCC tissues, and its expression level correlates with tumor stage, grade, degree of differentiation, lymph node metastasis, and survival time in OSCC patients.It may serve as a biomarker to determine the prognosis of OSCC patients and assist doctors in identifying high-risk patients, facilitating early, active, and personalized treatment post-surgery.Additionally, the underlying molecular mechanism appears to influence OSCC cells' invasion, migration, and proliferation by positively regulating the collagen-dependent PI3K/AKT signaling pathway.In subsequent studies, we will further investigate the specific molecular interactions between P4HA2 and the PI3K/AKT signaling pathway in OSCC cells and analyze P4HA2's role and mechanisms in OSCC in greater depth to propose novel ideas and methods for treating OSCC. https://doi.org/10.1038/s41598-024-64264-5 https://doi.org/10.1038/s41598-024-64264-5

Figure 1 .Figure 2 .
Figure 1.Investigation based on database in terms of OSCC's P4HA2 expression and the correlation of the expression with pathological feature, and pathway enrichment of P4HA2.(A, B) Investigation using UALCAN, TIMER, and TCGA databases regarding the expression levels of P4HA2 in OSCC and normal tissues, analyzed separately.In contrast to the results in normal tissues, OSCC tissues exhibited higher P4HA2 expression.(C) We analyzed the potential functions of P4HA2 through the GO database.The data indicated that P4HA2 was notably enriched in the PI3K/AKT signaling pathway.(D) Investigation based on the UALCAN database of P4HA2 levels in relation to clinical stage, pathological grades, and lymph node metastasis.The data indicated that P4HA2 expression correlated closely with clinical stage, pathological grades, and lymph node metastasis.*:p less than 0.05, **:p less than 0.01, ***:p less than 0.001, ****:p less than 0.0001.

Figure 3 .
Figure 3. Detection of transfection efficiency of cells and the influence of P4HA2 expression on the OSCC cells' apoptosis and proliferation.(A) qRT-PCR and WB tests were used to measure the transfection efficiency of OSCC cells.(B) The CCK-8 test was used to measure the cell growth rates in the sh-P4HA2, sh-GFP, pcDNA3.1-p4HA2,and pcDNA3.1 groups of SCC-25 and SCC-9.The data indicated that P4HA2 could facilitate the proliferation of SCC-25 and SCC-9.(C) The cell cycle profile in sh-P4HA2, sh-GFP, pcDNA3.1-p4HA2,and pcDNA3.1 groups of SCC-25 and SCC-9 was investigated by flow cytometry.The data indicated that P4HA2 could affect the cell cycle by regulating the G1G0/S phase.(D) Apoptosis in the sh-P4HA2, sh-GFP, pcDNA3.1-p4HA2,and pcDNA3.1 groups of SCC-25 and SCC-9 was investigated through flow cytometry.The data indicated that inhibition of P4HA2 expression could accelerate cell apoptosis.(E) WB test was used to measure the protein expression of Cyclin D1 and survivin in the sh-P4HA2, sh-GFP, pcDNA3.1-p4HA2,and pcDNA3.1 groups of SCC-25 and SCC-9.The data indicated that P4HA2 knockdown suppressed the expression of Cyclin D1 and survivin proteins.Conversely, we observed opposite results when P4HA2 was overexpressed.N = 3. *:p less than 0.05, **:p less than 0.01, ***:p less than 0.001, ****:p less than 0.0001.

Figure 5 .
Figure 5. P4HA2 Regulates the PI3K/AKT Pathway of OSCC Cells in a Collagen-Dependent Manner.(A, B) A WB test was used to measure the protein expression of collagen I, collagen IV, PI3K, p-PI3K, AKT, and p-AKT in the sh-GFP, sh-P4HA2, pcDNA3.1-p4HA2,and pcDNA3.1 groups of SCC-25 and SCC-9.The data indicated that the expression levels of collagen I, collagen IV, p-PI3K, and p-AKT in OSCC cells were notably reduced in the sh-P4HA2 group, while those in the pcDNA3.1-P4HA2group were notably higher than those in the sh-P4HA2 group.The expression levels of PI3K and AKT did not change notably between the sh-P4HA2 group and the pcDNA3.1-P4HA2group.(C) The effect of a P4HA2 enzyme agonist on the PI3K/AKT signaling pathway was determined by a WB assay in OSCC cell lines.N = 3. *:p less than 0.05, **:p less than 0.01, ***:p less than 0.001, ****:p less than 0.0001.

Figure 6 .
Figure 6.Down-regulated P4HA2 expression reduced OSCC cell proliferation and migration in vivo.(A) Based on the in vivo fluorescence imaging system, the Pulmonary metastasis model was identified for the evaluation of the migration capacity; the fluorescence intensity was analyzed.Next, the lungs of mice were isolated.(B) H&E staining was applied for the analysis of mice lungs' pulmonary metastasis cells (×100).(C) Effects of lentivirus infection on tumorigenicity of cells in the respective group.(D) The respective group material of body weight and tumor volume of mice.(E) H&E staining was used to indicate the pulmonary metastasis cells of mice lungs and the nucleus atypia of tumors.N = 6.*:p less than 0.05, **:p less than 0.01, ***:p less than 0.001, ****:p less than 0.0001. https://doi.org/10.1038/s41598-024-64264-5